Mammals express a variety of distinct, sometimes tissue-specific, cytosolic fatty acid binding proteins (FABPs). Despite efforts to isolate, identify ligand specificity, structurally characterize, and provide in vitro correlative functional data, the physiological functions of the FABPs within the cell are unknown. Fatty acid/fatty acyl CoA binding, previously attributed only to the FABPs, is shared by at least two other cytosolic proteins, sterol carrier protein, SCP-2 and acyl CoA binding protein, ACBP. The primary objective of this application is to resolve the relative physiological function(s) of these proteins in fatty acid uptake/trafficking/metabolism. (1) The fatty acid/fatty acyl CoA ligand binding site of SCP-2 will be structurally identified and characterized and compared to that of ACBP, L-FABP and I-FABP. (2) The relative ability of the 13, 15 and 58 kDa forms of SCP-2, L-FABP, I-FABP and ACBP to influence key regulatory enzymes in fatty acid/fatty acyl CoA esterification will be determined. (3) The regulatory role(s) of fatty acid/fatty acyl CoA in determining 58 Kda SCP-x fatty acid oxidative activity will be examined. (4) Embryonic stem (ES) cells and/or L cell fibroblasts wherein SCP-2 forms, ACBP, L-FABP, or I-FABP are overexpressed/gene ablated will be constructed/obtained, and fatty acid uptake, cytoplasmic diffusion/trafficking, and metabolic targeting will be examined. (5) The intracellular location of the 3 forms of SCP-2, ACBP-, L-FABP and I-FABP, will be determined in overexpressed/gene ablated cells. This proposal focuses on yielding basic new information on the role of the 3 forms of SCP-2 in fatty acid uptake, trafficking, and metabolism relative to the role(s) of the other two fatty acid/fatty acyl CoA binding protein families, FABPs, and ACBP. The results should provide fundamental new insights on potential roles and interactions of SCP-2s, ACBP and FABPs in fatty acid metabolism and whether dysfunction of any of these proteins relates to fatty acid malabsorption and peroxisomal fatty acid metabolic disorders such as Refsum's Syndrome and peroxisomal disorders in humans.